The present invention relates to the field of high-throughput quantitative protein analysis and, more specifically, to novel reagents for use in such analysis.
Most approaches to quantitative protein analysis are accomplished by combining protein separation, most commonly by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), with mass spectrometry (MS)-based sequence or tandem mass spectrometry (MS/MS)-based sequence identification of selected, separated protein species.
S. P. Gygi, et al., Nature Biotech, 17:994–999 (October 1999) describes an approach to quantitative protein analysis based on a class of reagents termed isotope-coded affinity tags (ICAT), which consist of three functional elements: a specific chemical reactivity, an isotopically coded linker, and an affinity tag. The reagents described by Gygi utilize biotin as the affinity tag and rely upon biotin-avidin affinity binding to isolate the cysteine-containing peptides from the complex peptide mixture.
Although the ICAT approach has many advantages over the traditional 2D-PAGE/MS approaches, it does possess some intrinsic limitations. For example, ICAT adds a relatively large chemical moiety onto the cysteine-containing peptides and this functionality is very labile under collision induced dissociation (CID) condition and thus complicates the downstream data analysis. Non-specific binding is also a concern since the enrichment relies on non-covalent affinity binding between a protein (avidin) and the biotinylated peptides. Finally, the captured peptides are not readily eluted from the avidin beads with high recovery using MS-compatible conditions. Thus, there is a need in the art for additional reagents and methods for improving performance in quantitative mass spectrometric analysis of protein mixtures.